Project 2: Replication Domain Organization During hESC Differentiation Dalton, Stephen A. Specific Aims. Many cytological, genetic and biochemical definitions of higher-order chromosomal domains have been put forth, but none provides a property that delineates boundaries of domains with precision and in a manner that can be applied genome-wide. Hence, while the word "domain" is frequently used to describe properties of large, often multi-genie, units of chromosomes, there is no comprehensive definition of a chromosome domain. We demonstrate that the "replication domain" is a definable chromosomal unit, revealing replication as potentially the only chromosomal property that affords a comprehensive segmentation of the entire genome at the megabase level. Importantly, we have discovered that the boundaries of these domains are dramatically re-organized during mESC and hESC differentiation to create larger temporally consolidated domains in which replication timing correlates more closely with sequence properties of chromosomes. Hence, replication structure of chromosomes in ESCs is relatively free from constraints imposed by DNA sequences, defining a novel property of pluripotent cells. We propose to further investigate the biological significance of domain consolidation, its relationship to the 3D organization of chromatin in the nucleus and the mechanisms by which consolidation occurs using hESCs as a model system. The specific aims are as follows: Aim 1: Choreography of Replication Domain Boundaries During Lineage Commitment. In this aim, we will test the hypothesis that replication domain boundaries are characteristic of particular cell types. More specifically, we propose that smaller temporally distinct replication domains is a characteristic of stem cells, and that differentiation will be accompanied by a progressively more rigid relationship between isochore sequence composition and replication timing. To determine whether boundaries are lineage specific, we will examine changes in replication domain boundaries during differentiation of hESCs to independent germ layers, ectoderm and mesendoderm. To distinguish whether changes in replication domain boundaries occur in a single-step, for example during loss of pluripotence, or whether there is a continuum of re-organization as cell lineage choices become more restricted, we will examine the downstream lineages definitive endoderm and mesoderm, as well as mesoderm differentiated to smooth muscle. Finally, to understand the functional significance of domain consolidation, we will determine which genes are affected by consolidation and how they relate to cell lineage choices. Aim 2: Replication Profiling as a Novel Means to Characterize hESCs. Replication profiling provides a convenient comprehensive genome-wide identification method that may shed light on important relationships between cell types and pluripotent cellular states that are currently being debated in the literature. In this Aim, we will identify the domains that distinguish hESCs from their differentiated counterparts. We will examine regions of conserved synteny between mouse and human for evolutionary conservation of replication domain structure. Finally, we will use replication profiling to address the debate as to whether hESCs are more similar to mESCs or mouse epiblast-like cells. Aim 3: Spatial consolidation of chromatin during differentiation. Here we will determine how the consolidation of replication domains defined molecularly in Aim1 corresponds to the three-dimensional re-organization of these domains in the cell nucleus. Using in situ hybridization at different stages of differentiation, we will trace the sub-nuclear localization of replication domains relative to specific sub-nuclear compartments to ask whether domains that consolidate temporally also consolidate spatially and whether they acquire a more uniform overall level of compaction. Aim 4: Coordination of replication forks during domain consolidation. In this Aim, we will analyze the polarity of replication forks, replicon sizes and regions of replication origin activity on individual stretched DNA fibers from the consolidating domains to identify replicon clusters and the locations of their boundaries relative to the boundaries of replication domains defined molecularly. These experiments will begin to define the molecular mechanisms by which domain consolidation occurs.